1. Technical Field
The present invention relates, in general, to the molecular cloning and expression of a receptor protein, and, in particular, to the D.sub.1 dopamine receptor linked to the activation of adenylyl cyclase activity. The invention further relates to a cDNA sequence encoding the D.sub.1 dopamine receptor, to a recombinant DNA molecule that includes such a sequence and to cells transformed therewith.
2. Background Information
Dopamine receptors belong to a large class of neurotransmitter and hormone receptors which are linked to their signal transduction pathways via guanine nucleotide binding regulatory (G) proteins, and are amongst the most intensively studied neurotransmitter receptor systems in the brain. Pharmacological, biochemical and physiological criteria have been used to describe two subcategories of dopamine receptors, referred to as D.sub.1 and D.sub.2. Creese, Receptor Bio. Chem. and Methodology 8:1-245 (1987). D.sub.1 receptors have been classically defined as being linked to the stimulation of adenylyl cyclase activity and are coupled with the G, regulatory protein. Krebabian, Nature 277:93-96 (1979); Krebabian, Trends Pharmacol 7:96-99 (1986). In contrast, activation of D.sub.2 receptors results in various responses, including inhibition of adenylyl cyclase activity, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. Creese, Ann, Rev. Neurosci 6:43-71 (1983); Vallar, Trends Pharmacol 10:74-77 (1989); Lacey, J. Physiol. 392:397-416 (1987); Bigornia, J. Neurochem. 51:999-1006 (1988).
The molecular characterization of D.sub.2 dopamine receptors has been facilitated by the cloning of a cDNA encoding a rat D.sub.2 receptor. Bunzow, Nature 336:783-787 (1988). More recently, this receptor has been shown to exist as two protein isoforms which are derived from a single gene yet produced by alternative RNA splicing. Monsma, Nature 342:926-929 (1989); Giros, Nature 342:923-926 (1989); Selbie, DNA 8:683-689 (1989); Dal Toso, EMBO J. 8:4025-4034 (1989); Grandy, Proc. Natl. Acad. Sci. 86:9762-9766 (1989); Chio, Nature 343:266-269 (1990); O'Malley, Biochemistry 29:1367-1371. This splice variation occurs in a region of the receptor which may be involved in G protein coupling, suggesting that these receptor isoforms may activate different signal transduction pathways.
Evidence has also accumulated suggesting heterogeneity in the D.sub.1 category of dopamine receptors. D.sub.1 receptors in renal tissue have recently been described as stimulating phospholipase C activity independently from that of adenylyl cyclase. Felder, J. Pharm. Exp. Therap. 248:171-175 (1989); Felder, J. Biol. Chem. 264:8739-8745 (1989); Felder, Am. J. Physiol. 275:F315-F327 (1989). There has also been shown, using Xenopus oocyte expression experiments, that rat striatal mRNA encodes D.sub.1 receptors which are coupled to phospholipase C and Ca2+ mobilization in a cAMP-independent fashion. Mahan, Proc. Natl. Acad. Sci. 87:2196-2200 (1990). These data suggest that there may be multiple D.sub.1 receptors which are coupled to different signal transduction pathways or that a single, multifunctional D.sub.1 receptor exists.
Dopamine receptors are extremely important from a clinical therapeutic viewpoint as drugs which activate (agonists) these receptors are used to treat Parkinson's disease and related extrapyramidal disorders as well as hyperprolactinemia, whereas drugs which block (antagonists) dopamine receptors are used to treat schizophrenia and other mental disorders. Despite their clinical utility, one problem with the dopamine agonist and antagonist drugs currently available is that they have many side effects, like many other drugs which work through interacting with receptors. These side effects are predominantly due to a lack of receptor specificity. That is, the drug in use interacts not only with dopamine receptors but with other neurotransmitter receptors as well.
A major goal of clinical neuropharmacology and the pharmaceutical industry is the development of more highly selective drugs with even greater efficacy than those currently in use. Impediments to this process are the low abundance of dopamine receptor protein available to study in neural tissue and the lack of suitable homogeneous model systems of the receptors with which to screen drugs against.
A novel approach to the solution of this problem is to clone cDNAs encoding dopamine receptors, construct eukaryotic expression vectors containing these cDNAs and create a series of stably transfected mammalian cell lines which express functional dopamine receptors in high abundance. These cell lines, which would express a homogeneous population of dopamine receptors, can be used by the pharmaceutical industry or others to screen drugs and study the dopamine receptors using a variety of biochemical, physiological and pharmacological techniques. To accomplish this goal, we have isolated a cDNA encoding the rat D.sub.1 dopamine receptor subtype linked to the activation of adenylyl cyclase activity. This cDNA encoding the D.sub.1 receptor will be inserted into different eukaryotic expression vectors and used in the construction of various mammalian cell lines expressing this functional protein. The resulting D.sub.1 receptor-expressing cell lines can be used to investigate the affinities and efficacies of agonist and antagonist drugs with the D.sub.1 receptor using various techniques, such as radioligand binding and second messenger assays.